FIONA Lab Report Answer Key

Cy3-DNA experiment

2. Below are snapshots of the movies with and without imaging buffer at frame 1, 10, 20, 30, 40, 50 and 60

3. The number on the ND filter represents the extent to which the laser light is attenuated. The light

intensity will be reduced by 10ND. Thus, filters with ND off 0.2, 0.6, 1.0, 2.5 and 4.0 will reduce the light

intensity by 100.2, 100.6, 101, 102.5, 104.0, which are 1.6, 4, 10, 316, and 10,000 times. The ND filter reduces

light intensity of a large range of wavelength equally

4. T-50 is the buffer that maintains pH. PCD and PCA are the deoxygenating agents which removes

oxygen from the solution and reduces the rate of photobleaching of fluorophores. Trolox is a reducing

agent that helps reduce blinking of fluorophores

5. FIONA tutorial answers:

• When FaceAlpha value is smaller, is the figure getting more transparent or more opaque?

o The figure becomes more transparent when FaceAlpha value is smaller

• What is the PixSize when the ObjMag is 60x and AddMag is 1x? (Assume ActPix is 16000 nm).

o 266.7 nm

• What is the PixSize when the ObjMag is 100x and AddMag is 1.5x?

o 106.7 nm

• The greater the magnification, is the PixSize smaller or bigger?

o Smaller

• What is the CountToPhoton for Sub-zero camera at 1 MHz 16 bit readout rate, 4.5x Preamp setting, and

200 EM gain?

o 0.0213

• What is the CountToPhoton for Sub-zero camera at 1 MHz 16 bit readout rate, 4.5x Preamp setting, and

50 EM gain?

o 0.0852

• When EM gain is bigger, is the CountToPhoton bigger of smaller?

o Smaller

• What is the standard deviation in x and y of the fit in units of pixel number?

o σx = 1.21 pixels and σy = 1.16 pixels

• What is the center of fit in x and y in units of pixel number?

o Centerx = 15.53 pixels and Centery = 14.83 pixels

• What is the EdgeColor when then input is [0 0 0], [0 0 1], [0 1 0], [1 0 0], [1 1 1] and [1 1 0.5]?

o Black, blue, green, red, white and yellow

• Does the fitted graph fit well with the raw data?

o Yes, the graph fits well to the raw data. We can see this from the almost random residual

• What is the standard deviation in x and y of the fit in units of nanometer?

o σx =129.4 nm and σy =123.8 nm

• What is the center of fit in x and y in units of nanometer?

o Centerx = 1657 nm and Centery = 1581 nm

• What is the background noise in units of number of photon?

o 7.64 nm

• What is the number of photon collected for the spot? To your knowledge, what is the number of photon

needed in order to get 1 nm accuracy?

o 1171 photons. Around 10,000 to 15,000 photons are needed to get 1 nm accuracy

• How big is the error caused by photon noise, pixel error and background in the x direction? Which is

the biggest term?

o Values of each term before taking the square root: Photon noise error: 14.3 nm2. Pixel error:

0.809 nm2. Background noise error: 26.4 nm2. The biggest error term is from the background

noise

• What is the precision in x and y?

o The precision in x and y are 6.4 nm and 6.0 nm respectively

• If your photon number is 15000, what is your precision in x and y? Out of the three terms, which is the

term that gives the greatest error?

o The precision will be 1.15 nm in x and 1.10 nm in y. The biggest error comes from the photon

noise. (1.11 nm2), then background noise (0.16 nm2), followed by pixel error (0.06 nm2)

6. Here are some example points analyzed with FIONA algorithm

No Preview PSF with Gaussian fit Graph of residual Localization

accuracy

Number of

photons

Background

noise (b)

1

x = 7.43 nm

y = 9.93 nm 231.25 50.6

2

x = 8.19 nm

y = 10.17

nm

136.3 45.6

3

x = 7.83 nm

y = 8.29 nm 252.2 31.0

4

x = 8.88 nm

y =9.91 nm 193.5 35.7

5

x = 7.92 nm

y = 8.99 nm 206.9 28.7

7. Below is the photobleaching lifetime for experiment with and without imaging buffer. With imaging

buffer (PCA/PCD and trolox), Cy3 photobleaches after around 450,000 photons. Without imaging

buffer, Cy3 photobleaches after around 50,000 photons. There are close to 10 fold improvements in

photostability when PCA/ PCD and trolox are used in experiment.

Photobleaching lifetime is different from fluorescence lifetime. Fluorescent lifetime refers to the average

time a fluorophore spends in the excited state before emitting a photon. Photobleaching lifetime measures

the tendency of a fluorophore to photobleach and stop fluorescing. Upon excitation, a fluorophore

transition from ground state into singlet excite d state. At this state, the fluorophore can transition back to

the ground state and emit a photon. Occasionally, fluorophore in singlet excited state will transition into

triplet excited state. This process is facilitated by oxygen. The triplet state is relatively long-lived compared

to the singlet state, and this allows more time for the fluorophore to react with other molecules and

photobleach. The photobleaching lifetime is usually measured in number of photons, while fluorescent

lifetime has units of ns. For cy3, the photobleaching lifetime can be between 10,000 and 5,000,000,

depending on the environment it is in. The fluorescent lifetime of Cy3 is around 0.3 ns.

Kinesin walking experiment

1. The step-size histogram is shown below. The expected step-size is 8.3 nm, which is the distance

between two tubulin heterodimers. Here we get 7.9 nm, which is very close to the expected step-size.

We can collect more data to get better statistics for future experiments to get more accurate step-size.

We can also try to image at higher frame rate to be able to see small step-sizes better, or higher laser

intensity to get better localization accuracy. We can reduce the ATP concentration to let kinesin walks

slower too in order to get more photons or collect at higher frame rate.

2. Below is the dwell-time histogram. We expect the dwell time histogram to be exponential, so it should

be fitted with an exponential graph as shown on the right graph below. The dwell time constant in the

example below is 0.74 step/s. This means that kinesin will step around 1.35 times every second.

3. The table below lists the average velocity for each ATP concentration used in experiment. When fitted

to the Michaelis Menten curve, we get a Km of 17 ± 9 uM. This is the ATP concentration at which the

velocity is half of the maximum velocity. The maximum velocity Vmax in this case is 440 nm/s.

Conc of ATP Avg Velocity Standard error

400 nM 16.1 7.7

1250 nM 85.7 14.6

4000 nM 91.5 9.6

12.5 uM 141.0 16.4

40 Um 409.2 53.9

200 uM 428.6 48.3

0 50 100 150 200

0

100

200

300

400

500

Velocity (nm/s)

Conc (uM)

Model Hill

Equationy=Vmax*x^n/(k^n+x^n)

Reduced Chi-Sqr 6.77635

Adj. R-Square 0.79814

Value Standard Error

Velocity Vmax 441.41715 136.17464

Velocity k 17.21541 9.34561

Velocity n 1 0