Linking Carbon Saturation Concepts to Nitrogen Saturation ...
Guide to Fluorescence Microscopy - Emory University€¦ · Tips •Use HiLo’sor other saturation...
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Transcript of Guide to Fluorescence Microscopy - Emory University€¦ · Tips •Use HiLo’sor other saturation...
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Guide to Fluorescence MicroscopyHow to Take Good Data
Neil Anthony, PhD
Picture
Picture vs. Data
Data
Picture
Picture vs. Data
Data
RGB
‘Blue’
‘Green’
‘Red’
RGB RGB RGB(#,#,#) (#,#,#) (#,#,#)
RGB RGB RGB(#,#,#) (#,#,#) (#,#,#)
RGB RGB RGB(#,#,#) (#,#,#) (#,#,#)
64 62 18
55 33 4
5 4 2
141 124 57
115 93 33
27 43 24
66 70 91
79 56 79
100 86 93
https://www.youtube.com/watch?v=1Xd_sLPwPp8
8 bit
8, 12, 15, 16 bit
Bits & Bytesb & B
• Each bit can be 0 or 1: binary• 8 bits make up 1 byte• e.g. 10 Gb/s LAN cable or
3,000 KB file size
# bits # options
1 2
2 4
…
8 256
12 4096
15 32768
16 65536
N 2N
Picture vs. Datahttps://www.youtube.com/watch?v=nqEKjVm1U-8
Before During After
Sections…
•Before - Misc
• Labeling Specificity
• Coverslips
• Mounting Media
• Refractive Index
• Objective Lenses
#1.5 – Thickness: 0.17 mm ± 0.01 mm
#1.5H – Thickness: 0.17 mm ± 0.005 mm
Refractive Index Matching
https://www.abberior-instruments.com/products/expert-line/easy3d-module/
Refractive Index Matching
Structured Illumination Microscopy (SIM)
http://jcb.rupress.org/node/28770
Waters 2009, J. Cell Biol. Vol. 185 No. 7 1135–1148
Lambert & Waters 2016, J. Cell Biol. Vol. 216 No. 1 53–63
http://jcb.rupress.org/content/216/1/53
Navigating challenges in the application of
superresolution microscopy
Accuracy and precision in quantitative fluorescence
microscopy
If it were confocal …
1.33
1.515
1.515
1.44
1.45
1.4
1.42
1.44
1.52
1.3 1.35 1.4 1.45 1.5 1.55
Prolong Glass
Prolong Gold (48hrs)
Prolong Gold (24hrs)
Fluoromount G
Vectashield
75% Glycerol
Coverslip
Imersion Oil
Water
Refractive Index
Resolution
http://iopscience.iop.org/article/10.1088/1361-648X/aa7185
Image Brightness
(Fluorescence) ~𝜆 𝑁𝐴4
𝑀2
~0.5 𝜆
𝑁𝐴
Confocal
Resolution
0
100
200
300
400
500
600
700
800
0
200
400
600
800
1000
1200
1400
1600
10 20 40 60 100
Res
olu
tio
n (
nm
)
Fiel
d o
f V
iew
(FO
V)
Mag.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
10 20 40 60 100
Bri
ghtn
ess
(AU
)
Nu
mer
ical
Ap
ertu
re (
NA
)
Mag.
Lenses
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html
http://www.bdbiosciences.com/us/s/spectrumviewer
https://searchlight.semrock.com/
•Before• Know your spectra• Know your hardware• Both inputs and outputs
https://www.fpbase.org/spectra/
Before –Know Your Hardware
• Laser Lines• Filter Sets
• (excitation & emission)• Spectral Detectors
Inputs & Outputs
Inputs
Outputs
• Laser Power• Photobleaching• Photodamage• Fluorescence/molecular
saturation• Scan Parameters
• # of Pixels/Lines/Stacks• Speed• Averaging
• Channel Setup• Bleedthrough (Crosstalk)• Simultaneous/Sequential
• Hg Lamp / LED(s)• Filter set• Attenuator
• Exposure Time
• Detector Settings• Gain / Smart Gain / HV *• Detector saturation• Detector type• Offset
• Noise• Signal to Noise Ratio (SNR)
• Files• Save Raw Data only **
*
**
Multiple Interconnected Parameters
Trade Offs…
Saturation - No averaging- Single channel
- Low gain -> 500- Increasing laser power
1% 2% 4% 8%
16% 32% 64% 100%
Eyes, Monitors, & LUTs
8 bit display
0 -
255
Monitors, Eyes, & LUTs
Monitors, Eyes, & LUTs
TitleLine Profiles
The best way to ‘see’ your data
Noise
http://jcb.rupress.org/content/185/7/1135
𝑁 = 𝑁S2 +𝑁𝑅
2 + N𝑇2
NS= Shot Noise
NR= Read Noise
NT= Thermal Noise
Display Settings& SNR
0 -
4095
min
-m
ax
0.5% 8% 100%32%2%
Offset
high offset low offset full range
Offset
high offset low offset full range
Under-sample or Over-sample?
497 nm/px
256, 512, 1024, & 2048 px (1/2s, 2s, 8s, 32s)
40X 1.30 NAWF ~230nmCF ~150nm
~190nm
248 nm/px
124 nm/px 62 nm/px
Under-sample or Over-sample?
497 nm/px 124 nm/px
Consider Line-Line Spacing
Sample not properly imaged
PMTs vs. HyDs(GaAsP)
Raw Data
Stretched Data
Use Gain on Olympus and Smart Gain on Leica sparingly
• Very Sensitive• Low Noise
Resonant Scanning4 vol/sec7 z/vol
CCD, EMCCD, & sCMOS
ExposureGain
ExposureGainTemperature
Exposure
Color Cameras
https://www.olympus-lifescience.com/
vs
Monochrome+
Tips • Use HiLo’s or other saturation indicator LUT
• Use contrast, histogram (log), and line profile to inspect the data
• Consider positive or negative controls, and sample variance; always start with positive sample
• Thoughtful filenames and/or project (lif) grouping
• Keep settings constant – Apples to Apples; line averaging acceptable
• Experiment with parameters• Always ask!
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