Lab 3: Single-Molecule Fluorescence Microscopy
(TA: Yunshan Fan and Todd Anderson)
Objective:
In this lab, we will learn total-internal reflection fluorescence microscopy (TIRFM) as a
useful method for studying single molecules, basic setup of TIRF, data acquisition and data
analysis.
Instruments, Software, and Chemicals:
Olympus IX70 inverted fluorescence microscope, Andor iXon Ultra 897 EMCCD
Camera, Andor Solis Software, ImageJ, glass cover slip, and resorufin.
Procedure:
Single molecule fluorescence with different fluorophore concentrations
1) Make resorufin dilutions to 100 nM, 10 nM, 1 nM, 100 pM and 10 pM final
concentration from a provided stock solution of 1 µM.
2) Turn on the laser and microscope, start EMCCD control and imaging program.
3) Select the 60x/1.45 oil TIRF objective, and apply a drop of immersion oil on it.
Note: Make sure there are no small air bubbles in the immersion oil droplet. Otherwise,
you will trap the air bubbles in between the objective and cover glass in step (5). It will
result in strange illumination patterns, and make the fluorescence image worse.
4) Place the cover glass on the sample stage and navigate to the area of interest by
using the XY translators located on the right hand side of the stage.
Note: The cover glass has been pre-cleaned by using piranha solution to remove any
adsorbed fluorescent impurities. Use tweezers to pick up the cover glass to avoid any
unnecessary contaminations.
5) Turn the coarse adjustment knob to move the objective upwards, when the immersion oil
contacts the cover glass. Then turn the fine adjustment knob to move the focal plane on
the top surface of the cover glass until you can clearly see the single molecule
fluorescence image.
Note: 60x/1.45 oil TIRF objective has a very short depth of field (~400 nm). Please keep
an eye on the fluorescence image while turning the fine adjustment knob or you may over
turn and miss the focal point. Moving the focus to the right position and getting a clear
single molecule fluorescence image may take some time for beginners.
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6) Add a drop of the 10 pM resorufin solution onto the cover glass, then observe and capture
a series of fluorescence images. Save the raw images (as .sif file) and convert to tiff
format.
7) Repeat step(6) and test all the resorufin solutions with different concentrations.
8) Import the saved images to ImageJ for data analysis. Plot the fluorescence intensity or the
number of fluorescent bright spots as the function of resorufin concentrations.
Note: The total images collected in this lab may take up to 1 GB of space. Use a flash
drive if you want to keep the data for future analysis.
9) After experiment, use isopropanol (IPA) and lens paper to remove the immersion oil and
clean the objective.
Note: Be careful when using lens tissue to remove oil and do not put pressure on the
objective while removing the oil because any grit in the oil could scratch the lens. It is
better to use multiple sheets of tissue to gently remove the oil. Do not polish the objective
lens with dry lens paper.
Post-Lab Questions:
1) What is Total Internal Reflection Fluorescence (TIRF)? Why it is essential to use TIRF to
detect single molecule fluorescence? Please draw a schematic diagram and explain how
TIRF works to give single-molecule sensitivity.
2) Search in the literature and find the quantum efficiency, extinction coefficient, and
absorption cross-section for the resorufin molecule.
Appendix
1. (1). The recommended settings of Andor
SOLIS for single molecule fluorescence
imaging:
Acquisition Mode: Kinetic
Exposure Time: 0.2 sec
Kinetic Series Length: 200
Electron Multiplier (EM) Gain: Enabled
Electron Multiplier Gain Level: 300
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(2). Convert the images to tiff format: FileSave as…
2. Analyze single molecule fluorescence images by using ImageJ
(http://rsb.info.nih.gov/ij/download.html)
(1) Import image stack: A folder of images can be opened as a stack either by dragging
and dropping the folder onto the ‘ImageJ’ window or by choosing
FileImportImage Sequence…
(2) Adjust brightness and contrast: ImageAdjust Brightness\Contrast …
(3) Count the number of fluorescent bright spots
a. Subtract background: ProcessSubtract Background… Rolling ball radius:
5~10 pixels
b. Apply a smoothing filter (3×3 mean filter): ProcessSmooth
c. Adjust the threshold: ImageAdjust Threshold… Lower slider: adjusts the
maximum threshold value until only the fluorescent spots are bright.
d. Convert to Mask: ProcessBinaryConvert to Mask…, run it twice, you
should see an inverted image
e. Count the number of the fluorescent spots: AnalyzeAnalyze Particles…
f. Save the pop-up results as a xls file, the values in the Count column are the
number of the fluorescent spots for each image/slice in the stack.
(4) Measure the total fluorescence intensity for each image/slice in the stack:
ImageStacksPlot Z-axis Profile
(5) Repeat above steps to analyze all the images with different resorufin concentrations.
Be sure to use the same parameters (brightness, contrast, threshold, etc.) for each of
the images stacks! Plot the fluorescence intensity or the number of fluorescent bright
spots as a function of resorufin concentrations.
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3.
Resorufin
CAS# 635-78-9
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