MODULE 5: SINGLE MOLECULE FLUORESCENCE › sites › default › files › yalec... · ‡ Principles of Fluorescence Spectroscopy, Chapter: Introduction to Fluorescence. For single

CHEMISTRY335L–SPRING2017–Z.GANIM

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MODULE 5: SINGLE MOLECULE FLUORESCENCE This lab module builds upon the understanding of brightfield microscopy, Köhler illumination, and

the Abbe diffraction experiments to introduce new concepts relevant to single molecule

fluorescence experiments. The goals of this module are to construct an epi-fluorescence

microscope with single molecule sensitivity on top of the brightfield microscope from Module 4, to

collect fluorescence image trajectories of single colloidal semiconductor nanoparticles (quantum

dots), and to perform image analysis in MATLAB that characterizes quantum dot photophysics and

the point-spread function of the microscope.

I. Instrument Layout

The schematic for the microscope you will construct serves as an outline for the new concepts in

this lab module (Figure 1).

The above diagram will be familiar as most of the optics serve the same purpose as in Module 4.

An additional light source is shown and the CMOS (complementary metal-oxide semiconductor)

camera from Module 4 is replaced with a CCD (charge coupled device) camera, which images the

sample plane (no back focal plane imaging in this experiment). In analyzing increasingly

complicated optical schemes, it is useful to consider the illumination and image paths separately.

This is facilitated by the breakdown in Figures 2 and 3.

Figure 1. Schematic of the epi-fluorescence and brightfield microscope. Inset indicates different beam paths.

CHEMISTRY335L–SPRING2017–MODULE5

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A. Brightfield Microscope Figure 2 shows the beam path for the brightfield microscope, which is virtually identical to what you

built in Module 4. This microscope will include a more powerful objective lens, a Nikon CFN Plan

100x NA=1.45 oil-immersion objective. Recall that the numerical aperture, which is one measure of

how well a lens collects light, is defined as, 𝑁𝐴 = 𝑛𝑠𝑖𝑛 𝜃 ,(1)

where n is the index of refraction of the medium and θ is half of the collection angle for the

outermost rays collected by the objective. In air, the maximum attainable NA is approximately 1,

which would correspond to a collection angle of 180°. By introducing a higher refractive index

medium in the gap between the objective and sample (typically a fluid with refractive index similar

to water, 1.33, or oil, 1.52), numerical apertures around 1.5 can routinely be obtained. This

procedure adds complexity to the experiment, as the fluid must be carefully placed (and cleaned)

and care must be taken not to prevent damage to the objective and sample when approaching.†

B. Epi-Fluorescence Microscope For an introduction to the phenomenon of fluorescence, please see Principles of Fluorescence

Spectroscopy by J.R. Lakowicz, Chapter 1, Sections 1.1-1.4.‡ For understanding the experimental

† See also Handbook of optics Vol.1, Chapter 28: Microscopes and Handbook of Biological Confocal

Microscopy, Chapter: Objective Lenses for Confocal Microscopy ‡ Principles of Fluorescence Spectroscopy, Chapter: Introduction to Fluorescence. For single molecule

fluorescence, see Single Molecule Analysis: Methods and Protocols, Chapter 5: A Brief Introduction to

Single-Molecule Fluorescence Methods

Figure 2. Simplified schematic of the brightfield microscope beam paths from Figure 1.


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design, the key concepts are that laser excitation of a fluorophore within its absorption band results

in frequency-shifted emission, which is emitted in all directions. § Both of these features are

exploited to isolate the fluorescence signal from background in the experimental design shown in

Figure 3.

A green laser (520 nm) is used to excite a uniform field of view in the sample, which is facilitated by

a focusing lens placed f away from the back focal plane of the objective. A dichroic mirror** routes

the laser towards the sample; this optic is >98% reflective to wavelengths <594 nm and >95%

transmissive to wavelengths >610nm (see Figure 6). As the laser is incident on this mirror, it is

reflected towards the sample. Fluorescence that is emitted at longer wavelengths (i.e., towards

lower energy or red-shifted) will pass through the dichroic towards the detector. Any back-

scattered laser light (from the several glass surfaces or the sample) will be reflected away from the

detector. A tube lens creates the fluorescence image on the CCD. Additionally, since the excitation

laser may be orders of magnitude brighter than the single molecule fluorescence you will observe,

a longpass filter†† (see Figure 6) rejects any laser light that happens to leak through the dichroic.

One may wonder why the (silver) mirror is introduced in Figure 3. Would it not be simpler to mount

the laser with its optical axis perpendicular to the dichroic? Upon some thought it becomes evident

that in such an arrangement, it becomes extremely difficult if not impossible to position the laser

§ Caveats abound from this generality, and the astute reader is encouraged to consider the photophysics

underlying their breakdown from texts such as Lakowicz. ** Chroma Technology Corp, T565lpxr-UF1 †† Chroma Technology Corp., ET570lp

Figure 3. Simplified schematic of the fluorescence microscope beam paths from Figure 1.


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carefully enough to send the beam directly through the center of the objective. One mirror can be

used to align the beam through one point. For the beam to travel through an assembly of lenses, it

must be aligned not to one point, but through a line. Since two points define a line, two degrees of

freedom must be adjusted. This concept is illustrated in Figure 4.

Figure 4. Optical Alignment. Beginning with a nominally misaligned system (A), it is possible to use one degree of freedom (M2) to steer the beam towards the target (B). In many applications, it is not desirable to have the optical axis tilted, such as when aligning through a system of lenses (or the microscope objective in this experiment). An additional degree of freedom, such as translation or rotation of the mirrors allows for a straightforward procedure to reliably reach alignment conditions. (C) Using standard mirror mounts that allow for tip/tilt adjustment, the first and second degrees of freedom are iterated to align the beam to a new optical axis. For maximum experimental stability and safety, it is best for the beam to be centered on the mirror before proceeding. New optics may be added and adjusted such that the optical axis is maintained.


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II. Signal, Background, and Noise The signal we would like to measure, the fluorescence of a single molecule, is very weak, and

typically ranges from 10-1,000,000 photons emitted per second. The ideal experiment would reject

photons generated by other processes and detect the signal by converting this stream of incident

photons into a measurable current or voltage with sufficient time resolution (i.e., shutter speed,

frames per second) to observe all relevant timescales. The governing parameters in weak signal

experiments are the background and noise. Background results from photons incident on the

detector from extraneous factors, such as stray light in the room, fluorescence of the optics,

impurities in the sample, and red-shifted light due to Raman scattering. If the background was

perfectly constant, it could simply be subtracted by measuring a control sample.

Noise is defined as fluctuations in the measured signal that do not yield useful information about

the system. Noise is introduced in the detection step, but also arises from fluctuations in the

background. Noise is quantified as the standard deviation in the measurement of an idealized

constant signal. The relevant figure of merit is the signal-to-noise ratio,

𝑆𝑁𝑅 =𝑆𝜎0(2)

For this lab, the signal is given by the product of the number of fluorescent photons from the

sample incident on the detector, PFl, multiplied by the probability of a charge being generated per

photon (the detector quantum efficiency, QE),

𝑆 = 𝑄𝐸 ∙ 𝑃67(3)

There are several contributions to the noise. For detectors that rely on the photoelectric effect –

incident photons generate free electrons – the discrete nature of electrons and Poisson counting

statistics gives rise to shot noise. This noise is proportional to the square root of the total number

of electrons being counted from signal photons and background photons, PBkg,

𝜎09:; = 𝑄𝐸 ∙ 𝑃<:;=7 = 𝑄𝐸 ∙ 𝑃67 + 𝑃?@A (4)

Once electrons are generated, they must be read, such as by discharging a capacitor, and this

leads to readout noise, σRead. Readout noise can be reduced by allowing the pixels to collect more

photons before being read resulting in less readout events (increase the integration time).

Alternatively, a 2x2 or 4x4 block of pixels can be read out as if they were one large pixel

(compromise spatial resolution). Standard hardware on detectors allows for the operator to tune

these parameters and they are included in the MicroManager software used in this experiment.


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Another source of noise is due to dark current; this quantifies the fact that electrons are released

due to the finite temperature of the detector, and counted along with the signal. If dark current was

perfectly constant in time, it could simply be subtracted, but due to the considerations above, it

must fluctuate and therefore generate noise, σDark. Although dark current is typically less than 1 e-

/pixel/second, it is significant at low light levels, and it can be reduced by cooling the detector.

Since sources of noise combine in quadrature (the square root of the sum of squares), the signal to

noise ratio for this example would be,‡‡

𝑆𝑁𝑅 =𝑄𝐸 ∙ 𝑃67

𝑄𝐸 ∙ 𝑃67 + 𝑃?@A + 𝜎CD=EF + 𝜎G=H@F(5)

To obtain best image quality, take the opportunity to explore and optimize your signal to noise

ratio!

III. Colloidal Semiconductor Nanocrystals The fluorescent molecules under study in this experiment are small (~1-10nm) crystals of

semiconductors or quantum dots. In the past decade these materials have rapidly emerged with a

wide range of scientific and technological applications due to their amenability to solution

processing and luminescent properties. Figure 5 presents an overview of the structure for a series

of CdSe/ZnS quantum dots, which demonstrates the tunability of their emission with particle size.

This is further detailed in Figure 6, which shows the broad absorption spectra and large molar

extinction typical of quantum dots (compare to a prototypical small molecule organic dye, such as

rhodamine 6G with 116,000 M-1cm-1 at 530nm.)

You will investigate the fluorescence intermittency or blinking behavior of quantum dots, which was

first reported in 1996§§ and has yet to be satisfactorily explained. In this phenomenon, the quantum

‡‡ For more detailed treatments, please refer to Hamamatsu’s “A Guide to Choosing the Right Detector,” “A

visual guide to CCD vs. EM-CCD vs. CMOS,” and Andor’s “Sensitivity of CCD Cameras.”

§§ “Fluorescence intermittency in single cadmium selenide nanocrystals,” M. Nirmal, B. O. Dabbousi, M. G.

Bawendi, J. J. Macklin, J. K. Trautman, T.D. Harris, L. E. Brus, Nature 383, 802 - 804 (1996)


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dot is observed not to continuously fluoresce when under continuous illumination, but rather

stochastically switches between a fluorescent bright state and nonfluorescent dark state. This

limits the utility of quantum dots in biological tagging applications, where naturally it is undesirable

for the fluorescent tag to spontaneously become dark during particle tracking. Moreover, this

blinking does not obey the exponential dependence expected for first- or second-order chemical

rate kinetics.***

*** “Modified Power Law Behavior in Quantum Dot Blinking: A Novel Role for Biexcitons and Auger Ionization,”

J. J. Peterson, D. J. Nesbitt, Nano Lett., 2009, 9 (1), pp 338–345

Figure 5. (a) Diagram representing the composition of a CdSe/ZnS quantum dot showing the core, shell, coating, and targeting molecules. The overall size is about 15 to 20 nm. (b) Micromolar aqueous solutions of 525, 565, 585, 605, and 655 quantum dots (left to right) under ultraviolet (UV) illumination. (c) Electron microscopic appearance of the same quantum dots (left to right) spread on a thin carbon substrate and air-dried. (d) Higher magnification image. Bar = 20 nm. Reproduced from "The application of fluorescent quantum dots to confocal, multiphoton, and electron microscopic imaging," Deerinck TJ, Toxicol Pathol. 2008 Jan;36(1):112-6. Copyright © by Society of Toxicologic Pathology and licensed under fair-use policy.


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IV. Additional Safety Precautions In this lab you will be using a Class 3B fiber-coupled laser emitting 1-10mW at 520 nm. The optical

fiber coupling the light from the laser diode to the exit aperture is somewhat fragile and may break

when bent sharply. Additionally, the power supply driving the laser is capable of outputting enough

power to damage the diode; although a soft limit has been preprogrammed, please mind the

driving voltage and please review the data sheet in lab to estimate the output power.

The safety glasses that shield your eyes from stray laser light will prevent you from seeing the beam

during alignment. Therefore, it is permissible to align the laser at low power (~1 mW, at which it is

comparable in power to a Class 2 laser). While the laser is on and not currently being aligned or

when you are situated at beam height (e.g., bending down to pick something up, sitting at the

Figure 6. (left) Molar extinction of the Qdot series from Invitrogen. (right) Normalized fluorescence

spectra of the same Qdot series plotted along the detector quantum efficiency and transmission of

optical components used in the microscope described herein. (Chroma Technology Corp, T565lpxr-UF1, Chroma Technology Corp., ET570lp, Photometrics Retiga R3)


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desk), you are requested to wear the supplied safety glasses. To minimize the chance of exposure,

please remove all reflective accessories, such as rings and watches, during the lab period.

Of course, all of the safety precautions for optics from Module 4 apply for Modules 5 and 6. Be

extra mindful to block the beam whenever inserting or removing optics from the beam path and

when handling the extremely fragile dichroic mirror.

Due to their heavy metal composition, the CdSe/ZnS samples in this lab are potentially toxic, and

as when handling any lab chemicals, you are requested to wear protective gloves and dispose of

the samples in the designated area.

V. Day One – Build the Microscopes The microscope you will be building, shown in Figure 1 is a modified version of what you have

constructed in Module 4. Therefore, consider the key points of the detailed alignment procedure

given there- why was that order of alignment suggested? How would you construct this new

instrument? Due to your expertise in optics, the alignment procedure herein is less detailed.

Step 1. Align the tube lens and CCD camera as you did in Module 4. Use the MicroManager

software to acquire an image of an object at infinity. Note that the objective lens and camera are at

a fixed height. Make sure that the longpass filter is in place on the camera before proceeding to

protect the CCD from bright light exposure and direct reflections from the laser. (You should still be

able to see clear brightfield images with the filter in place.)

Step 2. Insert the dichroic mirror (carefully). Since no light is on the table, you can sight

directly down the optical axis to make sure that the dichroic mirror is situated at 45 degrees and is

aligned squarely in front of the CCD.

Step 3. Prepare an iris to the exact height of the front aperture of the objective. Put an iris

on the rail right in front of the objective where the sample would be. When you close the iris all the

way down, you should still see the small front aperture of the objective.


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Step 4. Insert the silver mirror as shown in Figure 1. Coarsely adjust its position and tilt. It

should be angled at 45 degrees and situated in line with the dichroic.

Step 5. Before turning on the laser, think about its beam path and add beam blocks to

prevent any stray light (such as the small leak through the dichroic) from leaving the

optical breadboard.

Step 6. Turn the laser on and adjust it to a low power. For alignment, you want the power to

be <1mW. Check the manufacturer’s sheet to see which current this corresponds to. To turn the

laser on:

a) Switch on the QCombo-6305 power supply†††

b) Turn the “Laser Enable” key to the “On” position

c) Switch on the TEC (thermoelectric cooler)

d) Verify that the driving current is 0mA and switch on the laser.

e) Gradually increase the driving current until you can visualize the green laser spot on a

white card. Be careful as this step can result in stray reflections. Consider that any

surface that the laser strikes can partially reflect the light and act as a weak mirror.

f) Always use a white card to see exactly where the laser is at all times and make use of

the beam blocks.

Step 7. Adjust the tip/tilt mount that the laser is on, the silver mirror, and the dichroic

mirror such that the laser path is as shown in Figure 7A. By adjusting these three mounts,

you should be able to put your iris anywhere in the beam path and the light should travel through.

This will guarantee that when the focusing lens and objective are inserted correctly, the light will still

be aligned. Note the procedure detailed in Figure 4 for aligning the beam.

Step 8. Block the laser and insert the focusing lens (Figure 7B) lens at the correct height.

When the lens height and tilt are adjusted correctly, the light should still travel through the

alignment iris. No adjustment of the optics from Step 10 should be necessary. Translating the lens

should adjust the beam diameter without translating it, otherwise try redoing steps 3 and 10.

††† Instrument manual available at the QPhotonics website.


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Step 9. Block the laser and insert the objective lens (Figure 7C) lens. Although the beam

will be weak after the objective, you should still be able to see that it is centered on your iris. The

objective should be situated near the end of the rail (close to the dichroic) to give you maximum

room for the Köhler illumination setup below. Adjust the focusing lens such that the laser is

focused very close to the back aperture of the objective. Because it is difficult to gauge exactly

where the back focal plane is, later you will finely adjust the focusing lens to get a crisp image. No

adjustment of the optics from Step 10 or 11 should be necessary.

Figure 7. Illustration of alignment steps 7-9.


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Step 10. Build the remainder of the brightfield microscope as in Module 4. Note that this

objective lens is on a stable 1.5” pedestal mount that allows for fine translation along two axes.

Step 11. Insert the grating test sample into the sample holder and do a “dry run” of how

you will bring the slide towards the objective without adding immersion oil. (That is, verify

that you can freely move the slide holder and no obstructions are in place.) Note where the

objective will meet the sample. Also note the surface area of the face of the objective where it will

meet the sample – this indicates how much immersion oil needs to be added.

Step 12. Add a drop of immersion oil on the slide directly above the location where the

objective will meet the sample and as it slowly drips down, move the slide in place to

meet the objective. Some tips:

a) Be mindful of immersion oil drips and spills, such as off of the applicator after

you’ve added a drop onto the slide, by oil spots on your gloves, or by tilting the

bottle. Clean any excess immersion oil off of non-optical surfaces with

isopropanol and Kimwipes.

b) Optical surfaces should be cleaned of immersion oil using lens paper only.

c) Minimizing dripping by wiping the applicator after dipping it into the oil.

d) Practice may be required to calmly move the sample towards the objective until

full contact is made with the oil droplet without directly crashing the objective

into the slide. Feel free to wipe away the oil and begin again.

e) Due to the viscosity of the immersion oil, you should not move the slide position

as quickly as you did in Module 4; moving the slide quickly will result in air gaps

between the slide and objective.

Step 13. Once the objective and slide are in immersion oil contact, you may adjust the

focusing using the fine threaded knob on the objective lens mount.

Step 14. Using the grating test sample, derive a pixel to micrometer conversion and

estimate the resolution of the microscope. Record this image to include in your lab write-up.

Remove the sample and clean the surfaces before continuing.


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Step 15. Drop cast a fluorescence test sample. The TA will supply a sample of 800 nm

polyethylene microspheres impregnated with fluorescent dye Rhodamine B, which can be

visualized using both brightfield and fluorescence microscopy. (Spherotech FP-0858-2)

• Note that the objective is designed to image through a cover glass (0.17 mm) and

not through a microscope slide (1 mm). As such these slides are fragile and can

easily break when being screwed into the slide holder mount. (Expect to break one.

It’s ok; your tuition covers it.)

• To drop cast a sample, add ~3 μL of the sample to the middle of the cover glass

and allow the solvent to evaporate. With a permanent marker, draw a circle around

where the sample was added to facilitate finding it later (and in the dark). Label the

slide with its contents.

Step 16. Image your test fluorescence sample with laser excitation. You may need to adjust

the focusing lens such that the laser is focused at the back aperture of the objective. Record this

image to include in your lab write-up.

Step 17. Image your test fluorescence sample with both microscopes. You should be able

to turn on the LED see a brightfield image, then turn off the LED, turn on the laser and see a wide-

field fluorescence image. (What does it mean if you only see localized fluorescence from one spot

in the sample?) This is a good time to stop and diagnose any problems in your alignment of the

laser. Record this image to include in your lab write-up.

Step 18. Acquire a short movie of your fluorescence test sample. You should notice that the

fluorescence from the test sample decays in time – the sample gets dimmer. Record a movie that

captures this effect for later analysis.

Feel free to continue into Day Two tasks if time permits.


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VI. Day Two – Collect and Analyze Data Step 1. Drop cast a sample of quantum dots on a thin slide and mount it. Beginning with

the brightfield image, you should be able to see out-of-focus spherical particles corresponding to

dust on the surface. Adjust the focusing until you are imaging near the surface. In fluorescence

mode, you should see an array of bright spots. You may need to scan around the sample to find

regions with isolated quantum dots.

Step 2. Adjust the acquisition time and region of interest to collect fluorescence image

stacks (movies) from the sample.

• Note the laser intensity (derived from the driving current) for each acquisition and

optimize the following parameters for optimal data quality. (see Figure 8)

1. Exposure time - Choose this to get good image quality that samples the

blinking behavior with sufficient time resolution.

2. Binning - Spreading the signal out over many pixels when spatial resolution

is not desired will just increases the noise and makes the file size bigger

3. Region of interest - Collecting unilluminated or uninteresting regions makes

file bigger and analysis slower. Select a region (3b) and click the ROI icon

(3a) to preview the data that will be saved (3c)

4. Click Multi-D Acq. to set the movie parameters

5. Number of Time Points - This parameter sets the total number of frames

collected in the movie

6. Interval - To capture slow timescales without the blurring induced by long

exposure times, include an interval between exposures

7. Summary - Before acquiring a movie, inspect the file statistics – duration

and file size estimate

8. Directory Root and Name Prefix - Make sure you are writing data to the

desired location. Save your data as an Image stack file for ease of analysis

9. Acquire!


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• Without sufficient consideration, images of the entire CCD with fast data acquisition

can easily generate gigabyes of data! Select the above parameters carefully.

• Collect three movies emphasizing the blinking behavior of quantum dots.

o You will analyze these data to get on/off time distributions

o To minimize noise, bin neighboring pixels such that your quantum dot image

occupies approximately one binned pixel.

• Collect three movies that characterize the sharpest image you can obtain of

quantum dots

o You will analyze these data to characterize the point spread function of the

microscope. Set the binning to 1 pixel (no binning). You can use a small

region of interest that only shows a few quantum dots.

Figure 8. Illustration of MicroManager image and movie parameter settings.


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Step 3. Analyze the data with the MATLAB file qdot_time.m present your findings about

quantum dot blinking. MATLAB software installed on the lab computer can be used to reduce

the data from image stacks into intensity trajectories, intensity histograms, and histograms of on/off

times. Compile a collection of graphs that you can refer to in your lab write-up, which displays

what you have learned. Compare the results from the single molecule quantum dots to the many-

particle fluorescence in the test sample.

Step 4. Analyze the data with the MATLAB file qdot_psf.m to measure the point spread

function of the microscope. With the quantum dot sample, you have measured fluorescent

images from sources that are nearly point-like with respect to the laser wavelength. A perfect

microscope would produce image features that reflect the nanocrystal sizes (~1-10nm). In

practice, there are several limiting factors that may include the diffraction limit analyzed in Module

4, instrument misalignment, image aberrations, and detector pixel size. Quantify the smallest

particle diameter you find using the MATLAB software.

Step 5. Analyze the bleaching behavior of the bulk-like fluorescent beads with the

MATLAB file bulk_time.m to measure the bleach rate. You should have these data from

your work in Day 1, but feel free to acquire another if you are not satisfied with the data quality.

(optional) Step 6. Analyze the data to measure an interparticle spacing with

superresolution. Despite the granularity of the detection apparatus, you can fit the point-spread

function to obtain its center with better resolution than the diffraction limit allows. Performing this

on two immobilized particles, in conjunction with the pixel to micrometer conversion you’ve

derived, can be used to measure the distance between two quantum dots with superresolution. To

do this, you may modify the MATLAB software used in Step 4.

(optional) Step 7. Compare the fluorescence properties of the commercial CdSe beads

with your CdS and CdSe nanoparticles grown on graphene sheets from Module 2. You

may use qdot_psf.m to measure the distance between two particles with superresolution and/or

use qdot_time.m to compare the blinking statistics of the homemade quantum dots with the

engineered particles from Invitrogen.


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Optics and Materials Parts List Part Description Part Number* Tube lens f = 200 mm, Ø25mm, Achromat AC254-200-A Objective lens CFN Plan 100x NA=1.45 oil-immersion

objective (Out of production, but similar to Nikon CFI Plan Apo Lambda 100X Oil)

Condenser lens f = 50 mm, Ø25mm, Achromat AC254-050-A Field lens f = 150 mm, Ø25mm, Achromat AC254-150-A Collimating lens f = 20 mm, Ø25mm Aspheric ACL2520 White light LED 6500K color temperature, 800 mW MCWHL5 520 nm Laser Single mode fiber coupled laser diode,

8-10mW @ 520+/-20nm, in 14-pin butterfly package with built-in monitor photodiode, thermoelectric cooler, thermistor, and FC/APC fiber pigtail

QPhotonics QFLD-520-10S

Laser collimation lens Fiber Collimation Package, 530nm, f = 11.16mm, FC/APC

F220APC-532

Laser Power supply Fiber-coupled laser diode controller including laser driver, temperature controller, 14-pin butterfly mount

QPhotonics QCombo-6305

Focusing Lens f = 150 mm, Ø25mm, Achromat AC254-150-A Silver mirror Protected Silver Mirror, reflectance >

94% @ 450 nm - 20 µm ME2-P01

Dichroic beamsplitter Ø2” 50/50 @λ=450-650 nm Chroma T565lpxr-UF1 Longpass filter f = 50 mm, Ø25mm, Achromat Chroma, ET570lp CCD Camera 2.8 megapixel, monochrome, 14 bit,

fan-cooled charge coupled device Photometrics Retiga-R3

Vertical grating sample Variable Line Grating Slide, 3" x 1" R1L3S6P Test fluorescence sample

0.7-0.9 µm polyethylene microspheres, rhodamine B impregnated

Spherotech FP-0858-2

Quantum dot sample Qdot® 625 CdSe/Zns nanocrystal surface-functionalized with streptavidin

Invitrogen Q22063

f =focal length, Ø=diameter, λ=wavelength *ThorLabs parts numbers given unless otherwise specified


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520nm Laser Specifications Sheet


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VII. Lab-write up questions

A) Please include all images specified in the Day 1 and Day 2 instructions.

B) Compare the intensity trajectory from your test sample (many particle fluorescence) to the

quantum dots (single particle fluorescence). How do they behave differently in time? What

are the diagnostic features of single molecule fluorescence?

C) When discussing the quantum dot blinking, note which statistics are indicated by the

histograms of on/off times. (e.g., exponential, Gaussian, Poisson, power law)

D) Calculate the width of your point-spread function and compare this to what you would

expect from the diffraction limit. Estimate how close two point sources can be before you

would be unable to distinguish how many there are.

E) Please list the sources of fluorescence background you observed in this experiment.

F) Do a brief literature search, perhaps beginning with the references cited in this manual, to

outline current hypotheses for the physical mechanism underlying quantum dot blinking.

G) (Optional) Present the results of any optional experiments.

IX. Acknowledgements I would like to acknowledge Daniel Franke and Prof. Moungi Bawendi for permission to adapt this

lab module and its original conception and as a part of the Undergraduate Research Inspired

Experimental Chemistry Alternatives Modules at the Massachusetts Institute of Technology.

Documents

MODULE 5: SINGLE MOLECULE FLUORESCENCE › sites › default › files › yalec... · ‡ Principles of Fluorescence Spectroscopy, Chapter: Introduction to Fluorescence. For single